U.S. patent number 4,771,127 [Application Number 06/892,846] was granted by the patent office on 1988-09-13 for nontoxic pseudomonas aeruginosa polysaccharide-tetanus toxoid and polysaccharide-toxin a conjugate vaccines.
This patent grant is currently assigned to Swiss Serum & Vaccine Institute Berne. Invention is credited to Stanley J. Cryz, Emil P. Furer.
United States Patent |
4,771,127 |
Cryz , et al. |
September 13, 1988 |
Nontoxic pseudomonas aeruginosa polysaccharide-tetanus toxoid and
polysaccharide-toxin a conjugate vaccines
Abstract
Polysaccharide-protein conjugates were synthesized utilizing
polysaccharide derived from hydrolyized Pseudomonas aeruginosa
lipopolysacharide covalently coupled to either tetanus toxoid or P.
aeruginosa toxin A, utilizing a spacer molecule and a coupling
agent. Conjugates produced in such a manner possess a molecular
weight of greater than 350,000, are nontoxic and non-pyrogenic, and
upon immunization of animals induced protective anti-LPS antibody
and antibody which neutralizes the lethal effect of tetanus toxin
or toxin A. The polysaccharide-tetanus toxoid conjugate and
polysaccharide-toxin A conjugate are safe and immunogenic when
parenterally administered to humans.
Inventors: |
Cryz; Stanley J. (Bolligen,
CH), Furer; Emil P. (Muri, CH) |
Assignee: |
Swiss Serum & Vaccine Institute
Berne (Berne, CH)
|
Family
ID: |
4271640 |
Appl.
No.: |
06/892,846 |
Filed: |
August 4, 1986 |
Foreign Application Priority Data
|
|
|
|
|
Sep 27, 1985 [CH] |
|
|
04199/85 |
|
Current U.S.
Class: |
424/194.1;
530/402; 530/405; 424/197.11; 424/260.1; 514/54; 530/403 |
Current CPC
Class: |
C07K
16/1203 (20130101); A61K 39/104 (20130101); A61K
39/0258 (20130101); Y02A 50/474 (20180101); Y02A
50/30 (20180101); A61K 2039/6037 (20130101) |
Current International
Class: |
A61K
39/104 (20060101); C07K 16/12 (20060101); C07K
017/00 (); A61K 039/104 () |
Field of
Search: |
;424/92
;530/395,402,403,405 ;514/547 ;536/55.1,1.1 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Seid et al., JBC 1981, pp. 7305-7310..
|
Primary Examiner: Kight; John
Assistant Examiner: Draper; Garnette D.
Attorney, Agent or Firm: Kile; Bradford E.
Claims
We claim:
1. An immunogenic conjugate, comprising (i) a P. aeruginosa
polysaccharide covalently linked through at least one of a hydroxyl
or carboxyl group of said polysaccharide, said polysaccharide being
essentially free of lipid A, to (ii) a tetanus toxoid carrier
protein.
2. An immunogenic conjugate, comprising (i) a P. aeruginosa
polysaccharide covalently linked through at least one of a hydroxyl
or carboxyl group of said polysaccharide, said polysaccharide being
essentially free of lipid A, to (ii) a toxin A carrier protein.
3. The conjugate according to claim 1 or 2 wherein said conjugate
has a molecular weight greater than 350,000.
4. The immunogenic conjugate according to claim 1 or 2 further
comprising a spacer molecule, wherein said polysaccharide is
covalently linked to said carrier protein through said spacer
molecule.
5. The immunogenic conjugate according to claim 4 wherein said
spacer molecule is water soluble adipic acid dihydrazide.
6. A P. aeruginosa immunogenic vaccine comprising said conjugate
according to claim 1 or 2 and a pharmaceutically acceptable
carrier, wherein said conjugate is present in an amount sufficient
to elicit an immunogenic effect.
7. The vaccine according to claim 6 wherein said vaccine is
nontoxic and nonpyrogenic.
8. The vaccine according to claim 6 wherein said vaccine is capable
of inducing antibodies to both said polysaccharide and said carrier
protein of said conjugate.
Description
BACKGROUND OF THE INVENTION
Pseudomonas aeruginosa (P. aeruginosa) is a leading cause of
life-threatening nosocomial infections, especially in a compromised
host. Human immunity to P. aeruginosa has been correlated with
humoral antibody to lipopolysaccharide (LPS) and toxin A, as
described in Pollack M. Huang A. I., Prescott R. K., Young L. S.,
Hunter K. W., Cruess D. F., Tsai C. M., "Enhanced survival in
Pseudomonas aeruginosa septicemia associated with high levels of
circulating antibody to Escherichia coli endotoxin core," J. Clin.,
Invest. 1983; 72: 1874-1881; Pollack M., Young, L. S., "Protective
activity of antibodies to exotoxin A and lipopolysaccharide at the
onset of Pseudomonas aeruginosa septicemia in man," J. Clin.
Invest. 1979; 63: 276-286; and Cross A. C., Sadoff J. C., Iglewski
B. H., Sokol P. A., "Evidence for the role of toxin A in the
pathogenesis of infection with Pseudomonas aeruginosa in humans,"
J. Infect. Dis. 1980; 142: 538-546.
Anti-LPS antibody has been shown to be highly protective against P.
aeruginosa infections in a variety of animal model systems, as
noted in Cryz S. J. Jr., Furer E., Germanier R., "Protection
against Pseudomonas aeruginosa infection in a murine burn wound
sepsis model by passive transfer of antitoxin A, antilastase, and
antilipopolysaccharide," Infect. Immun. 1983; 39: 1072-1079; Cryz
S. J. Jr., Furer E., Germanier R., "Passive protection against
Pseudomonas aeruginosa infection in an experimental leukopenic
mouse model," Infect. Immun. 1983; 40: 659-664; Kazmierowski J. A.,
Reynolds H. Y., Kauffmann J. C., Durbin W. A., Graw R. G. Jr.,
Devlin H. B., "Experimental pneumonia due to Pseudomonas aeruginosa
in leukopenic dogs: prolongation of survival by combined treatment
with passive antibody to Pseudomonas and granulocyte transfusions,"
J. Infect. Dis. 1977; 135: 438-446; and Pier G. B., Sidberry H. F.,
Sadoff J. C., "Protective immunity induced in mice by immunization
with high-molecular-weight polysaccharide from Pseudomonas
aeruginosa," Infect. Immun. 1978; 22: 919-925. However, attempts to
use native P. aeruginosa LPS as a vaccine have been hampered by a
high frequency of adverse reactions following immunization and the
need for numerous injections to evoke an optimal immune response,
as noted in Alexander T. W., Fisher M., "Immunization against
Pseudomonas infection after thermal injury," J. Infect. Dis. 1974;
130 (Suppl.): 152-158; Haghbin M., Armstrong D., Murphy M. L.,
"Controlled prospective trial of Pseudomonas aeruginosa vaccine in
children with acute leukemia," Cancer 1973; 32: 761-766; and Young
L. S., Meyer R. D., Armstrong D., "Pseudomonas aeruginosa vaccine
in cancer patients," Annals Int. Med. 1973; 79: 518-527.
As described in Liu P.V., "Extracellular toxins of P. aeruginosa,"
J. Infect. Dis. 130 (Suppl.): 594-599 1974, toxin A is the most
toxic product, on a weight basis, synthesized by P. aeruginosa.
Toxin A acts to inhibit eucaryotic protein synthesis by catalyzing
the transfer of the adenosine diphosphate-ribosyl (ADPR) moiety of
nicotinamide adenine dinucleotide onto eucaryotic elongation factor
2, as discussed in Iglewski, B.H., Liu, P.V. and Kabat, D.,
"Mechanism of Action of P. aeruginosa exotoxin A: ADP-ribosylation
of mammalian elongation factor 2 in vitro and in vivo," Infect.
Immun. 15: 138-144, 1977 and Ohman, D.E., Burns R.P. and Iglewski
B.H., "Corneal Infections in mice with toxin A and elastase mutants
of P. aeruginosa," J. Infect. Dis. 142: 547-555, 1980. Antitoxin A
antibody either passively administered or induced by active
vaccination with a toxin A toxoid has provided significant
protection against experimental P. aeruginosa infection. Additional
investigations have demonstrated a direct correlation between
antitoxic antibody and survival of patients from an episode of P.
aeruginosa bacteremia, as described in Cross, A.S., Sadoff, J.C.,
Iglewski, B.H., and Sokol, P.A., "Evidence for the role of toxin A
in the pathogenesis of human infection with Pseudomonas," J.
Infect. Dis. 142: 538-46, 1980 and Pollack M.S. and Young, L.S.,
"Protective activity of antibodies to exotoxin A and
lipopolysaccharides at the outset of P. aeruginosa septicemia in
man," J. Clin. Invest. 63: 276-86, 1979.
Although native P. aeruginosa LPS contains protective serotype
specific antigenic determinants, native LPS has been found to be
too toxic for use in humans as a parenterally administered vaccine.
Serotype specific antigenic determinants of P. aeruginosa are
contained within the O-polysaccharide (PS) region of the LPS
molecule, and although the PS can be isolated free of the toxic
lipid A moiety of the LPS, the PS are non-immunogenic, as noted in
Pier, G.B., Sidberry, H.F., and Sadoff, J.C., "Protective Immunity
induced in mice by immunization with high molecular weight
polysaccharide from P. aeruginosa," Infect. Immun. 22: 919-925,
1978 and Chester, I.R., Meadow, P.M., and Pitt, T.L., "The
relationship between O-antigenic lipopolysaccharides and
serological specificty in strains of P. aeruginosa of different
O-serotypes," J. Gen. Microbiol, 78: 305-318, 1973. In order to
induce a protective immune response to isolated PS, by the present
invention isolated PS is covalently coupled to either tetanus
toxoid or P. aeruginosa toxin A, which serve as "carrier proteins"
for the PS. The conditions used to couple toxin A to PS effectively
d-etoxify the toxin A molecule, thereby producing a PS-toxin A
toxoid conjugate. The PS-toxin A and PS-tetanus toxoid conjugates
are non-toxic, immunogenic and provide protection against
experimental P. aeruginosa infections.
SUMMARY OF THE INVENTION
In one embodiment, the present invention comprises a method of
producing an immunogenic P. aeruginosa polysaccharide-tetanus
toxoid conjugate vaccine having the steps of: deriving
polysaccharide from P. aeruginosa lipopolysaccharide; covalently
linking a spacer molecule to the polysaccharide; contacting the
polysaccharide-spacer molecule with tetanus toxoid and a
water-soluble carbodiimide coupling compound; and recovering from
the resultant mixture a polysaccharide-tetanus toxoid
conjugate.
In another embodiment, the present invention comprises a method of
producing a polysaccharide-toxin A conjugate having the steps of:
deriving polysaccharide from P. aeruginosa lipopolysaccharide;
oxidizing the polysaccharide; linking toxin A covalently to a
spacer molecule using a water-soluble carbodiimide as a coupling
agent; and contacting the toxin A-spacer molecule with oxidized
polysaccharide to form an immunogenic polysaccharide-toxin A
conjugate.
In another embodiment the present invention comprises an
immunogenic P. aeruginosa vaccine comprised of a
polysaccharide-tetanus toxoid conjugate.
In another embodiment the present invention comprises an
immunogenic P. aeruginosa vaccine comprised of a
polysaccharide-toxin A conjugate wherein the toxin A portion of the
conjugate is detoxified by contact with the spacer molecule.
DETAILED DESCRIPTION OF THE INVENTION
In the description of the present invention hereinafter, reference
is made to the following tables:
Table 1 presents the molecular weight, toxicity, pyrogenicity and
immunogenicity of P. aeruginosa, strain PA220, polysaccharide
derived from hydrolized LPS, tetanus toxoid, P. aeruginosa toxin A,
polysaccharide-tetanus toxoid conjugate and polysaccharide-toxin A
conjugate;
TABLE 1
__________________________________________________________________________
Characteristics of PS, toxin A, tetanus toxoid, PS-toxin A and
PS-tetanus toxoid conjugates PS-toxin A PS-tetanus Toxoid PS
Tetanus Toxoid Toxin A Conjugate Conjugate
__________________________________________________________________________
Molecular weight.sup.1 <70,000 150,000 66,000 >350,000
>350,000 Toxicity.sup.2 Nontoxic Nontoxic 0.2 ug Nontoxic
Nontoxic Pyrogenicity.sup.3 50 ug 50 ug ND.sup.4 50 ug 85 ug
Immunogenicity.sup.5 Non-immunogenic Immunogenic Immunogenic
Immunogenic Immunogenic (Anti-tetanus (Antitoxin (Anti-PS (Anti-PS
and IgG) A IgG) and anti- anti-tetanus toxin A IgG) toxin IgG)
__________________________________________________________________________
.sup.1 Determined by gel filtration over a column of AcA34. .sup.2
Determined by intraperitoneal injection into 18-20 g female NMRI
mice. Expressed as the mean lethal dose per mouse. Nontoxic
signifies tha a minimum of 50 ug of antigen administered
intraperitoneally resulted in no mortality. .sup.3 The highest dose
of antigen when administered to rabbits by the intravenous route
which resulted in a rise in body temperature .ltoreq. + 0.3.degree.
C. Expressed as ug/ml/kg body weight. .sup.4 ND = not determined.
.sup.5 Determined by immunizing groups of 3 rabbits with 50 ug of
each antigen per rabbit. Sera were analyzed for specific IgG
antibody by ELISA
Table 2 shows the protective capacity of P. aeruginosa PA220 LPS,
the polysaccharide-tetanus toxoid conjugate and the
polysaccharide-toxin A conjugate vaccine against fatal P.
aeruginosa burn wound sepsis in mice;
TABLE 2 ______________________________________ Protection against
fatal P. aeruginosa PA220 burn wound sepsis by immunization with
PA220 LPS, PS-tetanus toxoid conjugate and PS-toxin A conjugate
Immunogen.sup.1 LD.sub.50 .sup.2
______________________________________ None 20.sup. LPS 10.sup.6
PS-tetanus toxoid 10.sup.6 conjugate PS-toxin A 10.sup.6 conjugate
______________________________________ .sup.1 Mice each received 1
ug of antigen intramuscularly on days 0 and 1 and were challenged
subsequent to burn trauma on day 28 with graded doses of P.
aeruginosa PA220 in accord with the method described in Holder, I.
A.; Wheeler, R., and Mortie, T. C., "Flageller preparations from P.
aeruginosa: animal protection studies," Infect. Immun. 35: 276-80,
1982. .sup.2 LD.sub.50 = mean lethal dose. Calculated by the method
described i Reed R. J. and Muench H. A., "Simple method of
estimating 50 percent end points," Am. J. Hyg. 27: 493-97,
1938.
Table 3 presents reactions noted by human volunteers after
parenteral immunization with 100 ug of polysaccharide-tetanus
toxoid conjugate;
TABLE 3
__________________________________________________________________________
Reactions following vaccination with PS-tetanus toxoid vaccine
Local Reactions Systemic Reactions Immunization Pain Swelling
Redness Total.sup.1 Fever Malaise Headache Other.sup.2 Total.sup.1
__________________________________________________________________________
1 6 2 1 6/16 0 1 0 1 2/16 2 3 2 2 .sup. 6/15.sup.3 0 0 0 0 0/15
__________________________________________________________________________
.sup.1 Number of volunteers noting a reaction/total number of
volunteers. .sup.2 One vaccine experienced swollen lymph nodes at
48 h postvaccination. .sup.3 One subject did not receive a second
dose of vaccine.
Table 4 shows the anti-PA220 LPS immunoglobulin G response in
volunteers after immunization with the polysaccharide-tetanus
toxoid vaccine; and
TABLE 4 ______________________________________ Anti-PA220 LPS IgG
antibody response following immunization with PS-tetanus toxoid
conjugate Mean ELISA titer (range).sup.1 Nr. .gtoreq. Pre-
Post-immune.sup.3 4-fold increase immune.sup.2 14 days 28 days in
titer (%).sup.4 ______________________________________ 43 (14-128)
652 (39-3,200).sup.5 823 (47-4,400).sup.5 13/16 (82)
______________________________________ .sup.1 ELISA titers
determined as described in Cryz, S. J. Jr., E. Furer, and R.
Germanier, "Protection against P. aeruginosa infection in a murine
burn wound sepsis model by passive transfer of antitoxin A,
antielastase, and antilipopolysaccharide" , Infect. Immun. 1983;
39: 1072-1079. .sup.2 Preimmune = at time of immunization. .sup.3
Postimmune = days postimmunization. .sup.4 Determined by dividing
postimmune titer by preimmune titer. .sup.5 The mean postimmune
titer was significantly elevated (p < 0.01 as determined by
students ttest) as compared to preimmune titer.
Table 5 shows the ability of human immunoglobulin G antibody
elicited in response to vaccination with the polysaccharide-tetanus
toxoid conjugate to protect mice against fatal P. aeruginosa
sepsis.
TABLE 5 ______________________________________ Capacity of
passively transferred human IgG to prevent fatal P. aeruginosa
PA220 burn wound sepsis in mice Anti-LPS IgG Transferred.sup.(1)
IgG ELISA titer LD.sub.50 ______________________________________
None.sup.(2) -- <1.3 .times. 10.sup.1 Pre-immune.sup.(3) 73 1.3
.times. 10.sup.3(5) Post-immune.sup.(4) 1065 6.7 .times.
10.sup.5(6) ______________________________________ .sup.(1) Mice
received approximately 160 ug of human IgG intravenously in 0.2 ml
24 hours prior to challenge with P. aeruginosa PA220. .sup.(2) Mice
received 0.2 ml of sterile saline. .sup.(3) Prepared from equal
aliquots of serum taken from all volunteers prior to immunization.
.sup.(4) Prepared from equal aliquots of serum taken from all
volunteers 28 days postimmunization. .sup.(5) 95% confidence
interval compared to LD.sub.50 of mice which received no IgG.
.sup.(6) 95% confidence interval compared to LD.sub.50 of mice
which received no IgG or preimmune IgG.
Table 6 shows the composition of conjugates formed by covalently
coupling polysaccharides derived from various serotypes of LPS to
tetanus toxoid.
TABLE 6 ______________________________________ Composition of
polysaccharide-tetanus toxoid conjugates Serotype of polysaccharide
Conjugate composition (%) (IATS) polysaccharide.sup.1 tetanus
toxoid.sup.2 ______________________________________ 1 27 73 2 17 83
3 22 78 4 22 78 6 43 57 7 39 61 10 40 60 11 21.5 78.5 16 32 68
______________________________________ .sup.1 Quantitated by the
phenolsulfuric acid method of Dubois et al. .sup.2 Quantitated by
the method of Lowry et al.
Table 7 shows the composition, molecular weight, ADPR-transferase
ctivity and pyrogenicity of a PS-toxin A conjugate vaccine
administered to human volunteers:
TABLE 7 ______________________________________ Characteristics of
PS-toxin A conjugate vaccine ADPR- Composition (%)* transferase
Pyrogenicity** protein carbohydrate M.sub.r.sup.+ activity
(.mu.g/kg) ______________________________________ 70.2 29.8
>350,000 ND*** 50 .mu.g (2 .times. 10.sup.6)
______________________________________ *Values shown are on a
weight basis. .sup.+ Determined by filtration over Sephacryl S500.
**ND = nonedetected. ***When administered intravenously to rabbits,
50 .mu.g of vaccine per kg body weight evoked <0.5.degree. C.
increase in temperature.
Table 8 presents reactions noted by human volunteers following
immunization with PS-toxin A conjugate:
TABLE 8 ______________________________________ Reactions noted
following immunization with PS-toxin A conjugate Immu- Local
reactions Systemic reactions niza- swell- red- itch- head- tion
pain ing ness ing fever chills malaise ache
______________________________________ 1 1 0 0 0 0 0 1 0 2 4 2 1 1
0 0 2 2 ______________________________________
Table 9 shows the immune response of human volunteers to
vaccination with PS-toxin A conjugate:
TABLE 9 ______________________________________ Immune response to
vaccination with PS-toxin A conjugate vaccine Mean .mu.g IgG/ml
Immune (Nr. .gtoreq. fold rise/total).sup.+ Peak Immunizing
response Day** fold dose* to 0 14 42 rise
______________________________________ 81.25 toxin A 0.61 19.9
(4/10) 22.5 (6/10) 37 LPS 9.3 108.7 (7/10) 63.3 (7/10) 11.6 162.5
toxin A 3.2 45.5 (4/10) 69.7 (9/10) 21.5 LPS 3.8 73.8 (7/10) 81
(9/10) 21.3 ______________________________________ *Volunteers were
immunized on day 0 and day 28. Subjects vaccinated with 81.25 .mu.g
of conjugate received 25 .mu.g of OPS and 56.25 .mu.g of toxi A
protein. Subjects vaccinated with 162.5 .mu.g of conjugate received
50 .mu.g of OPS and 112.5 .mu.g of toxin A protein. .sup.+
Indicates number of volunteers presenting with a 4fold or greater
rise in specific IgG compared to preimmunization levels. **Relative
to the time of immunization (day 0).
Table 10 shows the ability of human antitoxin IgG elicited in
response to vaccination with PS-toxin A conjugate to neutralize the
toxic effect of toxin A:
TABLE 10 ______________________________________ Ability of PS-toxin
A conjugate vaccine to elicit toxin A-neutralizing antibody
Immunizing dose* Mean .mu.g toxin A neutralized (.mu.g PS) per ml
of serum (range) ______________________________________ 81.25
pre-immune.sup.+ <0.312 post-immune** 3.1 (<0.312-19.9)
mean-fold rise*** 9.8 nr. .gtoreq. 2-fold rise 5/10 nr. .gtoreq.
4-fold rise 3/10 162.5 pre-immune.sup.+ <0.312 post-immune** 2.9
(<0.312-1.248) mean-fold rise*** 9.2 nr. .gtoreq. 2-fold rise
9/10 nr. .gtoreq. 4-fold rise 6/10
______________________________________ *Volunteers immunized with
81.25 .mu.g of conjugate received 25 .mu.g of OPS and 56.25 .mu.g
of toxin A protein. Volunteers immunized with 162.5 .mu.g of
conjugate received 50 .mu.g of OPS and 112.5 .mu.g of toxin A
protein. .sup.+ At time of immunization. **For serum collected at
day 42. ***Mean postimmune neutralizing capacity divided by mean
preimmune neutralizing capacity.
Table 11 presents the composition of conjugates formed by
covalently coupling polysaccharide derived from various serotypes
of LPS to toxin A:
TABLE 11 ______________________________________ Composition of
polysaccharide-toxin A conjugates Serotype of polysaccharide
Conjugate composition (%) (IATS) polysaccharide.sup.1 toxin A.sup.2
______________________________________ 1 35.5 64.5 2 35.5 64.5 3
27.5 72.5 4 28.6 71.4 6 43 57 7 26.5 73.5 10 35.5 64.5 11 35.5 64.5
16 29.6 70.4 ______________________________________ .sup.1
Quantitated by the phenolsulfuric acid method of Dubois et al.
.sup.2 Quantitated by the method of Lowry et al.
In generating the data included herein P. aeruginosa strain PA220,
Habs serotype 6, supplied by Dr. B. Wretlind, Karolinska Institute,
Stockholm, Sweden, was used as the source of lipopolysaccharide
(LSP). It is to be understood other strains of P. aeruginosa could
be used as a source of LPS, such as presented in the following
table, though not necessarily with equivalent results:
______________________________________ Interna- tional Antigen
Typing System (IATS) Strain Designation Serotype Source
______________________________________ PA53 1 Walter Reed Army
Institute of Research, Washington, D.C. E576 2 Walter Reed Army
Institute of Research, Washington, D.C. 8505 3 Public Health
Laboratory Service, Colindale, London, England 6511 4 Public Health
Laboratory Service, Colindale, London, England ATCC 27318 16 M.
Fisher Parke Davis and Co., Detroit, Michigan ATCC 27317 7 M.
Fisher Parke Davis and Co., Detroit, Michigan ATCC 27316 10 M.
Fisher Parke Davis and Co., Detroit, Michigan ATCC 27313 11 M.
Fisher Parke Davis and Co., Detroit, Michigan
______________________________________
The strain PA220 was serotyped as immunotype 1 according to the
Fisher-Devlin Typing System described in Fisher M., Devlin, H. B.,
Gnabsik, F., "New immunotype schema for P. aeruginosa based on
protective antigen," S. J. Bacterial 1969; 98: 835-36 or type 6 by
the International Antigenic Typing System (Difco Laboratories,
Detroit, Mich.). Cultures for challenge experiments were grown as
previously described in Cryz, S.J., Furer, E., Germanier, R.,
"Protection against P. aeruginosa infection in murine burn wound
sepsis model by passive transfer of antitoxin A, antielastase and
antilipopolysaccharide," Infect. Immun. 1983, 39: 1072-79.
Nontoxic immunogenic conjugates were synthesized by covalently
linking polysaccharide containing serological determinants derived
from hydrolized Pseudomonas aeruginosa PA220 LPS to tetanus toxoid
and polysaccharide derived from P. aeruginosa ATCC 27317 in an
identical manner to P. aeruginosa toxin A. Adipic acid dihydrazide
was used as a spacer molecule. The coupling of toxin A to
polysaccharide in this manner results in the detoxification of
toxin A, in essence producing a toxin A toxoid. The conjugates
produced in this manner possess a molecular weight of greater than
350,000, are nontoxic and nonpyrogenic, and induce antibody to
both-the polysaccharide and protein carrier, which when
administered to animals are protective against experimental P.
aeruginosa infections.
The polysaccharide-tetanus toxoid conjugate is safe when
parenterally administered to human volunteers. Vaccination
engendered an anti-polysaccharide and an anti-tetanus toxin
antibody response. These antibodies were protective against fatal
P. aeruginosa infections when passively transferred to mice and
neutralized the toxic effect of tetanus toxin.
By the method of the present invention, LPS was isolated and
purified as described by S. J. Cryz, E. Furer and R. Germanier
(1984), "Protection against fatal P. aeruginosa burn wound sepsis
by immunization with lipopolysaccharide and high molecular weight
polysaccharide," Infect. Immun. 43: 795-799. LPS prepared in this
manner contained less than 1% (wt/wt) protein and less than 1%
(wt/wt) nucleic acids. Polysaccharide (PS) containing serospecific
antigenic determinants was derived from LPS by mild acid
hydrolysis. The PS was derived from LPS by placing the LPS in 1%
(vol/vol) acetic acid and heating for 1.5 hours at 100.degree. C.,
as described by D. T. Drewry, K. C. Symes, G. W. Gray and S. G.
Wilkinson (1975), "Studies in polysaccharide fractions from the
lipopolysaccharide of P. aeruginosa," Biochem J. 149: 93-106.
Although, as noted above, in the preparation described herein PS
was derived from LPS by treatment with acetic acid for 1.5 hours at
100.degree. C., it is to be understood that other conditions for
deriving PS from LPS are within the scope of the present invention,
such as treatment with acetic acid for between 1/2 hour to 48 hours
and in a temperature range of from 70.degree. C. to 100.degree. C.
The major portion of the toxic lipid A moiety which was cleaved
from PS by this procedure was removed by centrifugation. The
supernatant was retained and extracted three times with a solution
of chloroform:methanol (3:1) to remove residual lipid A. The
aqueous PS-containing phase was retained and concentrated by rotary
evaporation over reduced pressure. This material was
chromatographed over a column of AcA34 (LBK-Produkter, Bromma,
Sweden). Polysaccharide with a molecular weight of approximately
10,000-75,000 was in this way collected, and lyophilized.
PS was next oxidized to generate reactive aldehyde groups as
follows. PS was dissolved in distilled water to a final
concentration of 5 mg/ml. NaIO.sub.4 was then added to yield a
final concentration of 0.1 M. This mixture was then stirred for 2
hours at 22.degree. C. protected from light. At this time 0.53 ml
of ethyleneglycol for the purpose of reacting with residual
NaIO.sub.4 was added and stirring continued for 30 minutes at
22.degree. C. This material was then extensively dialyzed against
distilled water and in turn, lyophilized.
Toxin A was purified as described in the Cryz, Furer, Germanier
reference "Protection against fatal P. aeruginosa infection in a
murine burn wound sepsis model by passive transfer of antitoxin A,
antielastase and antilipopolysaccharide," noted above, except that
the production strain was a spontaneously isolated hyper-producer
of toxin A derived from P. aeruginosa strain PA103 (supplied by Dr.
B. Wretlind, Karolinska Institute, Stockholm, Sweden) termed
PA103-FeR. The final preparation consisted of greater than 95%
toxin A protein as determined by high pressure liquid
chromatography.
Adipic acid dihydrazide (ADH) was utilized as a spacer molecular to
facilitate the covalent linking of toxin A to oxidized PS. ADH was
linked to toxin A as follows. Toxin A was diluted to 5 mg/ml in
0.05 M NaPO.sub.4, pH 7.2. ADH (Fluka A. G., Buchs, Switzerland)
and 1-ethyl-3 (-3-dimethylaminopropyl) carbodiimide were each added
as a solid to yield a final concentration of 10 mg/ml of each
reagent. This mixture was in turn dialyzed against 0.05 M
NaPO.sub.4, pH 8 buffer, for a period of 72 hours at 4.degree. C.
This mixture was subsequently dialyzed against 0.5 M NaPO.sub.4, pH
8 buffer, for a period of 4 hours at 22.degree. C. The resulting
insoluble material was removed by centrifugation and the toxin A
product concentration adjusted to 5 mg/ml. This product is termed
toxin A-ADH.
Toxin A-ADH was covalently coupled to the oxidized PS in the
following manner. Toxin A-ADH (in 0.5 M NaP04, pH 8 buffer) was
adjusted to a concentration of 5 mg/ml and added to an equal amount
of oxidized PS (5 mg/ml). The components were mixed by stirring for
6 hours at 22.degree. C. At this time 3.1 ml of 0.25 M NaCNBH3 was
added, followed by stirring for 5 days at 22.degree. C. This
mixture was then dialyzed for a period of 24 hours against
phosphate buffer saline, pH 7.4, containing 0.02% merthiolate.
Insoluble material was removed by centrifugation and the mixture
chromatographed over a column of AcA34 (LKB-Produkter, Bromma,
Sweden). Fractions were collected and monitored for absorbance at
220 nm and 280 nm. The material which eluted in the void (Vo),
possessing a molecular weight of greater than 350,000 (which
exceeded that of the starting PS or toxin A-ADH) representing the
PS-toxin A conjugate, was collected and lyophilized. The conjugate
was composed of 27.5% PS of the same serotype as determined by the
phenol sulfuric acid method described by Dubois et al. [M. Dubois,
K. A. Giles, J. K. Hamilton, P. A. Rebers, and F. Smith (1956)
Anal. Chem. 28, 350-356] using PS as a standard, and 72.5% toxin A
as determined by the procedure of Lowry et al. [O. H. Lowry, N. J.
Rosenbrough, A. L. Farr and R. J. Randall (1951) J. Biol. Chem.
193, 265-275] using bovine serum albumin as a standard. It is to be
understood that NaCNBH3 was utilized in the above procedure for the
purpose of reducing Schiff's bases formed above, and other
compounds exhibiting similar properties, such as NaBH.sub.4 could
be used.
In accordance with the present invention, the synthesis of the
PS-tetanus toxoid conjugate procedes as follows.
Tetanus toxoid for human use (TE Anatoxine, Swiss Serum and Vaccine
Institute, Berne, Switzerland) was used as a starting material.
Tetanus toxoid contained in TE Anatoxine was purified by
ion-exchange chromatography on DEAE-cellulose (Pharmacia Fine
Chemicals, Uppsala, Sweden) followed by gel filtration over AcA44
(LBK-Produkter, Bromma, Sweden). The final preparation consisted of
greater than 90% tetanus toxoid.
PS was isolated from LPS by acid hydrolysis as described above.
However, PS was employed in the non-oxidized form in the
conjugation with tetanus toxoid. PS (100 mg) was dissolved in 9.2
ml distilled water. Cyanogen bromide (0.44 ml of a 160 mg/ml
solution) was added for the purpose of introducing reactive cyclic
immidocarbonate or cyanate ester groups into the polysaccharide and
the pH maintained for six minutes at 10.5 by the addition of 0.1 N
NaOH. The pH was then lowered to 8.6 by the addition of solid
NaHCO.sub.3 and 0.4 g of ADH was added to equal 5 mg/ml. This
mixture was stirred for 16 hours at 4.degree. C., followed by
dialysis for a period of 72 hours against water. The pH of the
solution was lowered to 4.8 by the addition of HCl. Tetanus toxoid
(100 mg) and 1-ethyl 3- (-3-dimethylaminopropyl carbodiimide (400
mg) were added and the mixture stirred for 4 hours at 22.degree. C.
Following extensive dialysis for a period of 48 hours against
phosphate buffered saline, pH 7.4, the mixture was chromatographed
over a column of AcA34. Fractions were collected and monitored for
total protein content and total carbohydrate content. The material
which eluted in the void column, (Vo) possessing a molecular weight
in excess of 350,000, which exceeds that of the starting PS or
tetanus toxoid representing the PS-tetanus toxoid conjugate, was
collected and lyophilized. The conjugate was composed of 47.3%
tetanus toxoid (as determined by the method of Lowry et al. using
bovine serum albumin as a standard) and 52.7% PS (as determined by
the phenol sulfuric acid method using PS as a standard).
In the preparation of the polysaccharide-tetanus toxoid conjugate
and the polysaccharide-toxin A conjugate described above, adipic
acid dihydrazide was utilized as a spacer molecule. If desired,
other spacer molecules such as dicarboxylic acid dihydrazides of
the following nature NH.sub.2 --NH--CO--(CH.sub.2).sub.n
--CONHNH.sub.2, where n=1-10, could be utilized in the preparation
of these conjugates though not necessarily with equivalent
results.
Characteristics of PS-toxin A and PS-tetanus toxoid conjugates
Immunogenic characteristics of the PS-toxin A and PS-tetanus toxoid
conjugates are shown in Tables 1 through 11. With specific regard
to Table 1, the conjugate vaccines both possessed a molecular
weight in excess of 350,000, which exceeded the molecular weight of
their respective starting constituents (i.e., PS and either tetanus
toxoid or toxin A). Toxin A was highly lethal for mice with a mean
lethal dose of 0.2 ug/mouse. However, covalent coupling of toxin A
to PS resulted in a marked reduction in toxicity. There was no
mortality among mice which received 200 ug of toxin A protein
conjugated to PS intraperitoneally. Thus, the method described
herein utilized to produce the PS-toxin A conjugate reduces its
toxicity by a minimum of 1000-fold, in effect resulting in a toxin
A toxoid. All antigens, with the exception of toxin A which was not
assayed for pyrogenicity due to its highly toxic nature, were
nonpyrogenic when intravenously administered to rabbits in contrast
to native P. aeruginosa PA220 LPS which was pyrogenic at a dose of
0.7 ug/ml/kg body weight). PS alone was found to be non-immunogenic
in rabbits. In contrast, the PS-tetanus toxoid conjugate and the
PS-toxin A conjugate were found to induce antibody to both the PS
and respective carrier protein moieties.
Summarizing the data shown in Table 1: the PS-toxin A conjugate and
the PS-tetanus toxoid conjugate were of a high molecular weight,
nontoxic, nonpyrogenic and induced a specific antibody response to
both the PS and carrier protein moiety. Furthermore, the
conjugation conditions utilized produced a safe and immunogenic
toxin A toxoid.
The ability of the PS-toxin A conjugate vaccine to prevent fatal
intoxication of mice challenged with purified toxin A was
determined in the following manner. Groups of mice received either
10 ug of PS-toxin A conjugate or buffer (control groups)
intramuscularly on days 0 and 14. Mice were challenged
intraperitoneally on day 28 with grade doses of toxin A. The mean
lethal dose for the control group was 0.2 ug/mouse, whereas that
for the immunized group 4.7 ug/mouse, demonstrating that
immunization with the PS-toxin A conjugate induces antibody capable
of neutralizing the in vivo toxicity of toxin A.
The ability of native LPS, the PS-tetanus toxoid conjugate and the
PS-toxin A conjugate to protect against experimental P. aeruginosa
burn wound sepsis is shown in Table 2. Immunization with LPS,
PS-tetanus toxoid conjugate or PS-toxin A conjugate were equally
effective at preventing fatal sepsis, increasing the mean lethal
dose for P. aeruginosa PA220 50,000-fold over that for the
unimmunized group.
Safety and Immunogenicity of PS-tetanus toxoid vaccine in
humans
The PS-tetanus toxoid vaccine was prepared as follows. PS-tetanus
toxoid conjugate in phosphate-buffered saline plus 5% (wt/vol)
lactose was passed through a filter (0.45 um pore size) and
aseptically aliquoted into vials (1 human dose per vial =100 ug of
conjugate). The material was lyophilized and sealed under aseptic
conditions.
Tests for sterility and general safety of the vaccine was performed
according to the procedure detailed under articles V.2.1.1 and
V.2.1.5 of the European Pharmacopoeia, 2d ed., Sainte Ruffine,
France: Maisonneuve, S.A. The conjugate utilized in this example
was composed of 47.3% protein and 52.7% carbohydrate and bound both
anti-PA220 LPS IgG and anti-tetanus toxoid IgG when used as an
immobilized antigen in an ELISA assay system. The vaccine was found
to be comparatively non-pyrogenic for rabbits. The minimal
pyrogenic dose for PA220 LPS was 0.7 ug/kg whereas that for the
vaccine was more than 100-fold higher (85/ug). The vaccine was
nontoxic for mice and guinea pigs when injected intraperitoneally
(100 ug/animal) as evidenced by no overt signs of illness and
normal weight gain curves. When analyzed by gel immunodiffusion,
the conjugate gave a line of identity with PA220 LPS and tetanus
toxoid when tested against anti-PA220 LPS or anti-tetanus
toxoid.
Healthy adult volunteers aged 16-59 years of both sexes received
100 ug of conjugate in 0.5 ml pyrogen free water subcutaneously in
the deltoid area on days 0 and 14. All reactions were recorded by
the volunteers on a control sheet for 5 days post-vaccination.
Venous blood samples were drawn at the time of vaccination and at
14 days and 28 days post-vaccination. The sera was collected and
stored at -20.degree. C. Immunoglobulin G (IgG) antibody titers
were determined by ELISA assay as previously described in S. J.
Cryz, Jr., E. Furer, and R. Germanier (1983), Infect. Immun. 39:
1072-1079.
Reactions to vaccination are detailed in Table 3. Approximately 40%
of the vaccinees noted a local reaction following either
immunization. In most instances symptoms persisted for 24 hours or
less. Only two systemic reactions were noted, both after primary
vaccination. One vaccinee experienced malaise 4 hours
post-immunization which lasted for 4 hours. A second subject
reported swollen regional lymph nodes draining the site of
inoculation. All symptoms were spontaneously resolved and did not
interfere with normal activities.
Immunization with the PS-tetanus toxoid conjugate resulted in a
significant (p<0.01) rise in the mean anti-PA220 LPS IgG titer
at 14 days and 28 days post-vaccination as compared to the mean
pre-immune titer. (Table 4). Over 80% (13/16) of the volunteers
responded with a significant (4-fold or greater) increase in
anti-PA220 LPS IgG titer. Immunization with the conjugate also
resulted in an increase in tetanus toxin neutralizing antibody
titer. Two serum pools were made by combining an equivalent amount
of serum from each individual at the time of immunization
(pre-immune pool) and 28 days post-immunization (immune pool). The
pre-immune pool possessed 2.7 international tetanus toxoid
neutralizing units per ml of serum, whereas the immune pool
possessed 6.2 units per ml of serum.
Anti-PA220 LPS IgG antibody evoked by vaccination of humans with
the PS-tetanus toxoid conjugate vaccine was found to be highly
effective at preventing fatal experimental P. aeruginosa sepsis
(Table 5). Passively transferred post-immune IgG was significantly
(p<0.05) more effective at preventing death than passively
transferred pre-immune IgG. Protection correlated with the
anti-PA220 LPS IgG titers possessed by the IgG preparations.
To summarize the data from Tables 3-5: the PS-tetanus toxoid
conjugate was found to be safe when parenterally administered to
humans, evoking only mild, self-limiting reactions in less than
half the subjects. The conjugates elicited a significant increase
in anti-PA220 LPS IgG titers in greater than 80% of vaccinees. This
anti-PA220 LPS IgG was found to be highly protective against fatal
experimental P. aeruginosa PA220 sepsis.
Safety and Immunogencity of PS-toxin A conjugate in humans
As noted previously, Table 7 presents various characteristics of
the PS-toxin A conjugate administered to human volunteers. The
conjugate was composed of 70.2% toxin A (protein) and 29.8%
carbohydrate (PS). The molecular weight of the conjugate was
approximately 2.times.10.sup.6 It was devoid of ADPR-transferase
activity and was nonpyrogenic. It is important to note that the
toxic nature of toxin A is believed to be due to its
ADPR-transferase activity, and the fact that the conjugate lacks
this activity explains its nontoxic nature. As shown in the table
below, toxin A is rendered non-toxic through the covalent linkage
of toxin A with the ADH spacer molecule.
______________________________________ Toxicity of toxin A and
toxin A-ADH Antigen Mean lethal dose.sup.1
______________________________________ toxin A 0.2 ug toxin A-ADH
>10 ug.sup.2 ______________________________________ .sup.1
Expressed as ug of antigen per mouse administered
intraperitoneally. .sup.2 More than 50% of the animals
survived.
Twenty healthy adult volunteers were vaccinated with the
polysaccharide-toxin A conjugate vaccine. As shown in Table 8,
reactions to immunization were mild in nature, consisting primarily
of local discomfort at the injection site. All symptoms were
spontaneously resolved of their own accord and did not hinder the
normal activity of the vaccinees.
The immune response of the volunteers to vaccination is shown in
Table 9. The conjugate, at both doses tested, was capable of
eliciting immunoglobulin G (IgG) antibody to both the PS component
(represented by anti-LPS IgG) and the toxin A component
(represented by antitoxic IgG). Peak fold rises in IgG to toxin A
were from 21.5-fold to 37-fold (for the 162.5 ug dose and the 81.25
ug dose, respectively). The mean fold rise in IgG to LPS ranged
from 11.6-fold to 21.3-fold (for the 81.25 ug dose and the 162.5 ug
dose, respectively). At 42 days post-immunization, the
seroconversion rate (.gtoreq.4-fold rise) ranged from 60% to 90%
for both toxin A and LPS at the 162.5 ug dose.
As presented in Table 10, antitoxin IgG elicited in response to
vaccination with the PS-toxin A conjugate was found to be capable
of neutralizing the toxin effect of toxin A.
In addition, as shown in Tables 6 and 11, the procedure described
therein can be utilized to synthesize conjugates composed of
various serotypes of PS using either toxin A or tetanus toxoid as a
carrier protein. These conjugates of different polysaccharide
serotypes can be combined to form polyvalent polysaccharide-tetanus
toxoid and polysaccharide-toxin A vaccines of various
polysaccharide serotype combinations.
PS-tetanus toxoid conjugate and PS-toxin A conjugation in vaccine
use
The usefulness of PS-tetanus toxoid conjugate and PS-toxin A
conjugate as a vaccine is apparent from the data presented in
Tables 1-11. Both conjugates are nontoxic and nonpyrogenic.
Immunization with either conjugate evokes serotype-specific
anti-LPS antibody which is highly protective against experimental
P. aeruginosa burn wound sepsis. The PS-toxin A conjugate described
herein also engenders a toxin A neutralizing antibody response in
addition to eliciting protective anti-LPS IgG antibody, and the
protective capacity of specific antitoxin A antibody has been
demonstrated. As described previously, passive transfer of an
antitoxin A globulin protected mice against lethal intraperitoneal
infections with P. aeruginosa, and passively administered antitoxin
A antibody protected mice which were burn-traumatized from
subsequent P. aeruginosa infections. In addition, the ability of
the PS-toxin A conjugate to evoke an immunogenic response to humans
to both LPS and toxin A has been demonstrated.
In humans, a P. aeruginosa bacteremic episode has been shown to
result in an increase in serum antitoxin A titers. High titers of
antitoxin A antibody at the onset of P. aeruginosa bacteremia
correlate with an increased survival rate and it has been shown
that those patients who survived a bacteremic infection with a
toxin-A producing strain of P. aeruginosa had sub stantially higher
mean peak antitoxin A titers (25.8.+-.5.5 ug/ml) than patients who
died from such infections (4.6.+-.2.0 ug/ml) as described in A. S.
Cross, J. C. Sadoff, B. H. Iglewski, and P. A. Sokol (1980), J.
Infect. Dis. 142: 538-546. The mechanisms by which anti-LPS and
antitoxin A mediate their protective capacity based upon survival
rates for humans suffering from P. aeruginosa bacteremia appear to
be different and synergistic. Therefore, since the PS-toxin A
conjugate elicits 2 types of protective antibodies, anti-LPS and
antitoxin A, this conjugate provides a higher degree of protection
against P. aeruginosa infection than a corresponding vaccine which
would induce only anti-LPS antibody.
While a preferred embodiment of the invention has been described
herein, it will be obvious to those skilled in the art that various
changes and modifications may be made without departing from the
spirit of the invention as defined in the following claims.
* * * * *